Summary

The Aα3 and Aβ3 genes, which together constitute the A42 mating type factor of Coprinus cinereus, were isolated from a cosmid genomic library by walking 50 kb, a map distance of 0.5 units, from the closely linked metabolic gene pab-1. Cosmid clones having A gene function were identified by transformation into compatible A6 (α2β2) and A5 (α1β1) host cells where either α3 or β3 was expected to elicit the A factor — regulated development of unfused clamp cells. DNAs were digested with various enzymes before transformation in order to identify the smallest fragments containing an active α3 or β3 gene. Two non-overlapping fragments were identified as containing the α3 and β3 genes respectively. Southern hybridisation analyses showed that these two cloned genes had no detectable sequence homology, and that there was little or no hybridisation to the α and β gene alleles that constitute the A5 and A6 factors. α3 and β3 were shown to be less than 2.0 kb apart and embedded in a DNA sequence extending over 9.0 kb which was unique to our A42 strain and may contain a third A factor gene.

Summary

Using synthetic oligonucleotide probes, we cloned genomic DNA sequences encoding an α-amylase gene from Aspergillus niger var. awamori (A. awamori) on a 5.8 kb EcoRI fragment. Hybridization experiments, using a portion of this cloned fragment to probe DNA from A. awamori, suggested the presence of two α-amylase gene copies which were subsequently cloned as 7 kb (designated as amyA) and 4 kb (amyB) HindIII fragments. DNA sequence analysis of the amyA and amyB genes revealed the following: (1) Both genes are arranged as nine exons and eight introns; (2) The nucleotide sequences of amyA and amyB are identical throughout all but the last few nucleotides of their respective coding regions; (3) The amyA and amyB genes from A. awamori share extensive homology (≥98% identity) with the genes encoding Taka-amylase from A. oryzae. In order to test whether both amyA and amyB were functional in the genome, we constructed vectors containing gene fusions of either amyA and amyB to bovine prochymosin cDNA and used these vectors to transform A. awamori. Transformants which contained either the amyA- or amyB-prochymosin gene fusions produced extracellular chymosin, suggesting that both genes are functional.

Summary

Wa have cloned and characterized the cDNA coding for a major component of cellulase, endoglucanase (FI-CMCase), produced by Aspergillus aculeatus. The cDNA was isolated from a A. aculeatus cDNA library using synthetic oligonuceotide mixtures that correspond to the internal amino acid sequence of the mature FI-CMCase protein. Nucleotide sequence analysis of the cloned cDNA insert revealed a 711 bp open reading frame that encoded a protein of 237 amino acid residues. The primary structure of FI-CMCase deduced from the nucleotide sequence of cDNA agreed with that found by amino acid sequencing of peptide fragments obtained by digestion with several proteinases and cyanogen bromide cleavage. There may be a signal peptide sequence of 16 amino acid residues at the N-terminus. The molecular mass of the mature protein calculated from the cDNA is 24002 daltons, which compares favorably with molecular mass estimates of purified FI-CMCase obtained from SDS-PAGE (25000 Da). No distinct homology was found between the amino acid sequence of FI-CMCase and known cellulase sequences of other microorganisms. This study is the first example of cDNA cloning of an endoglucanase from the genus Aspergillus.

Summary

A HeLa cell cDNA library on a yeast expression vector was used to complement auxotrophic markers of Saccharomyces cerevisiae. Clones complementing the ade2-101 mutation harbor a 1.5 kb poly(A)+ tailed insert with a 425 amino acid open reading frame hybridizing with two human mRNAs of 1.5 kb and 3.1 kb. Its 5′ half is homologous to Bacillus subtilis SAICAR synthetase (E.C.6.3.2.6.) and its 3′ terminal half corresponds to the catalytic subunit of Escherichia coli and B. subtilis AIR carboxylase (E.C.4.1.1.21). In agreement with these homologies, pADE2H1 clones complement both ade1 and ade2 mutants of S. cerevisiae, as was also recently reported for a 3.1 kb cDNA isolated from human hepatocytes.

Summary

We have developed a rapid, simple transformation procedure for intact cells of Hansenula polymorpha. It is a modification of the LiAc method and can yield 104–105 transformants/μg DNA. The use of stationaryphase cells, a high cell density per plate and a hear pulse at 50°C for 10 min are among major modifications of the original method. We have also found that, within the XhoI-SalI chromosomal fragment of Saccharomyces cerevisae containing the LEU2 gene, a sequence exists which supports autonomous replication of plasmid moleculles in H. polymorpha.

Summary

Orotate phosphoribosyl transferase (OPRTase) catalyses the transformation of orotate to OMP in the pyrimidine pathway. In the yeast Saccharomyces cerevisiae, the URA5 gene is known to encode this enzyme activity. In this paper we present the cloning and sequencing of a yeast gene, named URA10, encoding a second OPRTase enzyme. Comparison of the predicted amino acid sequences between URA5 and URA10 genes shows more than 75% similarity. These sequences have also been compared to those of Escherichia coli, Podospora anserina, Sordaria macrospora and Dictyostelium discoideum. Remarkable similarities in the primary structure of these proteins have been found. Gene disruption experiments revealed that URA10 gene expression is responsible for the leaky phenotype of a ura5 mutant. Assays of OPRTase activity in extracts from ura5 and ura10 mutants indicate that the URA10 product contributes only 20% of the total activity found in wild type cells.

Summary

We have cloned the Cephalosporium acremonium pyr4 gene by cross-hybridization with the equivalent gene from Neurospora crassa, the closest relative from which this gene is available. The C. acremonium pyr4 gene complements an E. coli pyrF mutant lacking orotidine-5′-phosphate decarboxylase (OMPdecase), and most probably does not contain introns. Maxicell analysis in E. coli shows that it encodes a 46 kDa polypeptide. The C. acremonium OMPdecase contains a highly conserved pentadecapeptide characteristic for this category of enzyme. Extensive sequence comparison suggests an important role of this region in enzymatic activity.

Summary

A P. stipitis cDNA library in λgt11 was screened using antisera against P. stipitis xylose reductase and xylitol dehydrogenase, respectively. The resulting cDNA clones served as probes for screening a P. stipitis genomic library. The genomic XYL2 gene was isolated and the nucleotide sequence of the 1089 bp structural gene, and of adjacent non-coding regions, was determined. The XYL2 open-reading frame codes for a protein of 363 amino acids with a predicted molecular mass of 38.5 kDa. The XYL2 gene is actively expressed in S. cerevisiae transformants. S. cerevisiae cells transformed with a plasmid, pRD1, containing both the xylose reductase gene (XYL1) and the xylitol dehydrogenase gene (XYL2), were able to grow on xylose as a sole carbon source. In contrast to aerobic glucose metabolism, S. cerevisiae XYL1-XYL2 transformants utilize xylose almost entirely oxidatively.

Summary

Using the previously cloned Aspergillus niger N756 pectin lyase D gene as a probe, the corresponding pelD gene has been isolated from a genomic library of the loboratory strain A. niger N400. This gene encodes PLD, previously described as PLI, which is one of the two major pectin lyases isolated from the commeriial pectinase preparation Ultrazym®. Heterologous hybridization of the A. niger N400 genomic library with the pelD gene led to the isolation of another five genes: pelA, B, C, E, and F. These genes differ in their hybridization patterns with probes containing either the entire pelD gene, or 5′ or 3′ parts thereof. By partial sequencing, and expression in an A. niger transformant containing multiple copies of the pelA gene, we show that this gene, which hybridizes strongest with the pelD gene, encodes the other major pectin lyase from Ultrazym®, PLII.

Summary

Mating pheromones, known as a and α-factors, arrest the division of cells of opposite mating types, α and a respectively, in Saccharomyces cerevisiae. I have cloned the DAC2 gene, which is required for both pheromone-induced division-arrest and cell-fusion during conjugation. The constructed dac2::LEU2 null mutation leads to defects in both pheromone-induced divisionarrest and cell-fusion during conjugation; it also suppresses the growth defect caused by the gpal mutation (a mutation in the α subunit of the S. cerevisiae G protein). These results indicate that DAC2 may be the same gene as FUS3, which was recently isolated by Elion et al. (1990) as a gene essential for cell-fusion during conjugation. The dac2::LEU2 null mutant also showed morphological alterations in response to mating pheromones. I show here that the DAC2 product plays an essential role in both the division-arrest signalling pathway of the yeast pheromone response and in cell-fusion during conjugation.